Reagent for streptococcal anti-esterase assay

A reagent for the determination of an antibody against an esterase from pathogenic streptococci, which comprisesreagent 1: an esterase (a) from pathogenic streptococci,reagent 2: a protein (b) which is capable of binding to an antibody (d) against the esterase (a) and is bound to an insoluble carrier, andreagent 3: a reagent (c) for measuring an activity of the esterase (a),and a method for the determination of an antibody against an esterase from pathogenic streptococci. The reagent and method of the present invention are useful for diagnosis of various diseases caused by pathogenic streptococcal infections.

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Description

The present invention is illustrated by the following Examples but is not limited thereto. In the Examples, "%" is % by weight, unless specified otherwise.

EXAMPLE 1

Preparation of esterase (a) (antigen):

A culture medium (pH 7.4, 10 liters) composed of 0.025% of glucose, 0.3% of beef heart infusion, 2% of polypeptone, 0.2% of sodium chloride, 0.2% of dried yeast and 0.4% of disodium hydrogen phosphate was inoculated with Streptococcus pyogenes SS 379, type A-40, and the medium was subjected to stationary culture at 37.degree. C. for 16 hours. The resulting culture broth was centrifuged to remove the cells. To the filtrate was added solid ammonium sulfate to give 60% saturation, and the mixture was centrifuged at 6,000.times.g for 20 minutes. The resulting precipitate was collected and dissolved in a small amount of 0.02M phosphate buffer (pH 7.0), and the mixture was dialyzed against the same buffer solution for 8 hours and lyophilized to give a powder (1.2 g) containing type A-I esterase.

In the same manner as described above, Streptococcus pyogenes 69882, type A-49 and Streptococcus sp H36B, type B-I-b were cultivated, and there was obtained each powder containing type A-II or type B esterase, respectively.

EXAMPLE 2

Preparation of bacterial cell walls containing "protein-A" (immuno-adsorbent):

A 100 liter fermentor containing a culture medium (pH 7.0, 50 liters) composed of 2% of Bactocasamino acid (manufactured by Difco), 2% of yeast extract, 0.7% of sodium lactate, 2% of sodium glycerophosphate, 0.02% of magnesium sulfate, 0.001% of manganese sulfate, 0.32% of ferric sulfate, 0.32% of sodium citrate, 0.4% of potassium dihydrogen phosphate and 6.25% of disodium hydrogen phosphate was inoculated with Staphylococcus aureus Cowan I, and the medium was cultivated for 20 hours at 37.degree. C. (aeration rate, 50 liters/min.; agitation speed, 150 r.p.m.). The resulting culture broth was centrifuged to isolate the cells. The cells thus obtained were suspended in water and were disrupted, and the resulting mixture was washed with 1M aqueous sodium chloride solution (twice) and with water using a centrifuge, and lyophilized to give cell walls containing "protein-A" (58 g).

EXAMPLE 3

Preparation of insoluble antibody (immuno-adsorbent):

An anti-human immunoglobulin rabbit antiserum (50 ml), which was prepared by immunizing rabbit with human immunoglobulin, was diluted with 0.1M potassium phosphate buffer (pH 7.0, 150 ml), and thereto was gradually added a saturated aqueous ammonium sulfate solution (200 ml). The mixture was stirred under ice-cooling for 20 minutes and then centrifuged at 11,000.times.g for 20 minutes. The resulting precipitate was collected and dissolved in 0.1M potassium phosphate buffer (pH 7.0) to make a total volume of 100 ml. To the solution was gradually added a saturated aqueous ammonium sulfate solution (43 ml), and the mixture was stirred under ice-cooling for 20 minutes and again centrifuged. The supernatant fluid was separated and thereto was gradually added a saturated aqueous ammonium sulfate solution (39 ml), and the mixture was stirred under ice-cooling for 20 minutes and then centrifuged. The resulting precipitate was collected and dissolved in 0.02M potassium phosphate buffer (pH 7.0, 100 ml). The mixture was dialyzed against 0.02M phosphate buffer (pH 7.0, 10 liters) in a dialysis tube overnight. The dialysis was repeated twice.

The dialyzed inner solution was adjusted with the same buffer solution to give a protein concentration of 1 g/50 ml. The resulting mixture was mixed with an aqueous suspension (20 ml) containing cell walls of Lactobacillus plantarum ATCC-8014 (1 g, an insoluble carrier) and 1M sodium acetate buffer (pH 5.0, 10 ml). To the mixture was gradually added with stirring 0.8% aqueous glutaraldehyde solution (120 ml) and the mixture was stirred an for additional 2 hours. The reaction mixture was centrifuged at 6,000.times.g for 10 minutes. The precipitate was collected and suspended in 0.1% bovine serum albumin (BSA) - 0.9% NaCl - 0.04M potassium phosphate buffer (pH 7.0, 500 ml) and subjected to the sonication at 19 KC for 30 seconds, and the resulting mixture was centrifuged. The resulting precipitate was collected and added to the same buffer as used above (100 ml), and the mixture was again sonicated and centrifuged. The procedure was repeated twice, and the resulting precipitate was collected and added to the same BSA-NaCl-potassium phosphate buffer as above (100 ml), and the mixture was sonicated again to give a suspension containing bacterial cell wall-bound immunoglobulin rabbit antibody (an insoluble antibody).

The suspension of an insoluble antibody thus obtained was used for the determination of anti-esterase antibody in the following Examples.

EXAMPLE 4

Determination of anti-type A-I esterase antibody:

reagent 1: A solution (10 ml) which was prepared by dissolving the type A-I esterase (50 mg) obtained in Example 1 in water

reagent 2: A suspension (10 ml) which was prepared by suspending the cell walls of Staphylococcus aureus Cowan I (750 mg) obtained in Example 2 in water

reagent 3-1: 0.3 mM-DTNB-0.05M Tris-HCl buffer (pH 8.0)

reagent 3-2: Substrate solution, 10 mM S-acetylthiophenol in ethanol

reagent 3-3: Acetone

buffer solution I: 0.02M Phosphate buffer (pH 7.0)

buffer solution II: 0.02M Tris-HCl buffer (pH 8.0)

Sera containing the anti-esterase antibody (d) obtained from patients infected by hemolytic streptococci (two boys of 6 and 4 years old) were used as the test material. The sera were heated at 57.degree. C. for 30 minutes.

Each serum was diluted to 20 fold with buffer solution I, and the diluted serum (0.1 ml) was added to a test tube, and thereto was added Reagent 1 (antigen) (50 .mu.l), and the mixture was incubated at 37.degree. C. for 10 minutes to proceed to antigen-antibody reaction. To the reaction mixture was added reagent 2 (immuno-adsorbent) (0.1 ml), and the mixture was again incubated at 37.degree. C. for 30 minutes. The reaction mixture was washed with buffer solution II twice and centrifuged. The resulting precipitate was collected and suspehded in reagent 3-1 (coloring agent) (1 ml) and thereto was added Reagent 3-2 (substrate) (40 .mu.l), and the mixture was incubated at 37.degree. C. for 30 minutes. After adding reagent 3-3 (enzymatic stopping reagent) (2 ml), the reaction mixture was centrifuged at 1,700.times.g for 10 minutes. The absorbance of the resulting yellow supernatant fluid was measured at 412 nm in a cell (light path: 1 cm). As a result, the serum from the boy of 6 or 4 years old showed a value of anti-type A-I esterase antibody of 380 or 320, respectively.

The absorbance multiplied by a factor of 1000 was used as the value of antibody (1000.times.A.sub.412 /5 .mu.l serum/30 min. at 37.degree. C.).

EXAMPLE 5

In the same manner as described in Example 4 except that the type A-II esterase obtained in Example 1 was used as reagent 1 and the insoluble antibody obtained in Example 3 was used as reagent 3, two sera obtained from patients infected by hemolytic streptococci (girl of 7 years old and boy of 4 years old) were assayed. As a result, the sera showed a value of anti-type A-II esterase antibody of 852 and 662, respectively.

EXAMPLE 6

In the same manner as described in Example 4 except that the type B esterase obtained in Example 1 was used as reagent 1, a serum obtained from a patient infected by hemolytic streptococci (girl of 8 years old) was assayed. As a result, the serum showed a value of anti-type B esterase antibody of 924.

EXAMPLE 7

Differential determination of various types of antibody:

In the same manner as described in Example 4 except that either type A-I, type A-II or type B esterase was added as reagent 1, a serum obtained from a patient infected by hemolytic streptococci (girl of 7 years old) and a serum obtained from a non-infected healthy boy (6 years old) were assayed, by which each of three anti-esterase antibodies was differentially measured. The results are shown in Table 1.

                TABLE 1                                                     

     ______________________________________                                    

              Antibody value                                                   

     Test Material                                                             

                Type A-I    Type A-II Type B                                   

     ______________________________________                                    

     Serum of girl                                                             

                970         190       1250                                     

     infected by                                                               

     hemolytic                                                                 

     streptococci                                                              

     Serum of non-                                                             

                 71          55        32                                      

     infected boy                                                              

     ______________________________________
EXAMPLE 8

Determination of anti-type A-I esterase antibody:

Sera obtained from patients infected by hemolytic streptococci (girl of 5 years old and boy of 6 years old) were used as the test material. The sera were heated at 57.degree. C. for 30 minutes.

Each serum was diluted to 20 fold with buffer solution I as used in Example 4, and the diluted serum (0.1 ml) was added to a test tube, and thereto was added reagent 2 (immuno-adsorbent) (0.1 ml) as used in Example 4, and the mixture was incubated at 37.degree. C. for 30 minutes. The resulting precipitate was collected by centrifugation and washed with buffer solution II as used in Example 4 with a centrifuge. To the resulting precipitate was added Buffer solution I (0.1 ml) as used in Example 4, and the mixture was stirred and thereto was further added reagent 1 (50 .mu.l) as used in Example 4, and the mixture was incubated at 37.degree. C. After 10 minutes, the resulting precipitate was washed with buffer solution II as used in Example 4 with a centrifuge. The activity of esterase in the precipitate was measured in the same manner as in Example 4. As a result, the serum of the girl of 5 years old or the boy of 6 years old showed a value of anti-type A-I esterase antibody of 720 or 430, respectively.

EXAMPLE 9

Determination of anti-type A-I esterase antibody:

A serum obtained from a boy (5 years old) infected by hemolytic streptococci was heated at 57.degree. C. for 30 minutes. The serum was diluted to 20 fold with buffer solution I as used in Example 4. The diluted serum (0.1 ml) was added to a test tube, and thereto was added reagent 1 (50 .mu.l) as used in Example 4 and reagent 2 (0.1 ml) as used in Example 4, and the mixture was incubated at 37.degree. C. for 10 minutes. The resulting precipitate was treated in the same manner as described in Example 4, and the antibody value was measured likewise. As a result, the serum showed an antibody value of 475.

EXAMPLE 10

Comparison of the methods in Examples 4, 8 and 9:

With respect to several sera obtained from several children patients infected by hemolytic streptococci as mentioned in Table 2, the anti-type A-I esterase antibody was measured in the same manner as described in Examples 4, 8 and 9, respectively. The results are shown in Table 2.

                TABLE 2                                                     

     ______________________________________                                    

              Method for measurement                                           

     Test material                                                             

                Example 4   Example 8 Example 9                                

     ______________________________________                                    

     Serum of girl                                                             

                100.0       89.9      92.0                                     

     of 4 years old                                                            

     Serum of girl                                                             

                100.0       92.3      90.9                                     

     of 5 years old                                                            

     Serum of boy                                                              

                100.0       87.5      93.7                                     

     of 5 years old                                                            

     Serum of boy                                                              

                100.0       98.2      88.5                                     

     of 4 years old                                                            

     Serum of girl                                                             

                100.0       90.5      95.2                                     

     of 6 years old                                                            

     ______________________________________                                    

      [Remark]:                                                                

      The value in the above table shows the relative antibody value when the  

      value in Example 4 is shown as 100.
EXAMPLE 11

Determination of anti-type A-I esterase antibody:

The serum obtained from a girl (8 years old) as used in Example 7 was diluted 2 fold successively with buffer solution I as used in Example 4, and values of the anti-type A-I esterase antibody were determined in the same manner as described in Example 4. As shown in Table 3, there was a linear relationship between the antibody values and dilution folds.

                TABLE 3                                                     

     ______________________________________                                    

     Degree of dilution                                                        

                1/16     1/8    1/4     1/2  1/1                               

     Antibody value                                                            

                58       121    245     487  975                               

     ______________________________________
EXAMPLE 12

Correlation between the value of anti-type A-I esterase antibody and ASO value:

With respect to sera obtained from 102 child patients infected by hemolytic streptococci, the value of anti-type A-I esterase antibody was determined in the same manner as described in Example 4 and the data were compared with the value of an antibody against Streptolysin O (ASO value) determined by the method of Rantz-Randall [cf. Proc. Soc. Exp. Biol. Med., 59, 22 (1945)]. The correlation of both data is shown in the accompanying FIG. 1.

Claims

1. A reagent kit for the quantitative determination of an antibody against an esterase from pathogenic streptococci in human blood serum, comprising:

(a) a predetermined quantity of an esterase from pathogenic streptococci, said predetermined quantity being at least immunochemically equivalent to the quantity of said antibody to be determined;
(b) a predetermined quantity of a protein-A, said protein-A being able to bind non-specifically to said antibody to be determined and being bound to an insoluble carrier, said predetermined quantity being at least immunochemically equivalent to the quantity of said antibody to be determined; and
(c) a predetermined quantity of a substrate reagent that measures esterase activity, which is selected from the group consisting of S-acetylthiophenol, naphthylacetate and nitrophenylacetate, and said predetermined quantity being sufficient to measure quantitatively the esterase in an esterase-anti-esterase antibody-insoluble protein-A complex.

2. The reagent kit of claim 1, wherein said esterase is selected from the group consisting of a type A-I esterase, a type A-II esterase, the type B esterase, and a mixture thereof.

3. The reagent kit of claim 1, wherein said insoluble carrier is selected from the group consisting of bacterial cell walls, glass beads, and a hydrophilic, insoluble molecular-sieve chromatographic medium, made by cross-linking dextran.

4. The reagent kit of claim 1, wherein said protein-A bound to an insoluble carrier is bacterial cell walls containing a protein-A.

5. A method for the quantitative determination of an antibody against an esterase from pathogenic streptococci in human blood serum comprising:

(a) adding at least an immunochemical euqivalent of an esterase from pathogenic streptococci to human blood serum containing said antibody to be determined;
(b) adding to the mixture of step (a) at least an immunochemical equivalent of a protein-A, said protein-A being able to bind non-specifically to said antibody to be determined and being bound to an insoluble carrier to form an esterase-anti-esterase antibody-insoluble protein-A complex;
(c) separating said complex from the mixture of step (b) by centrifugation; and
(d) mesuring the activity of the esterase in said complex by using a substrate which is selected from the group consisting of S-acethylthiophenol, naphthylacetate and nitrophenlacetate.

6. A method for the quantitative determination of an antibody against an esterase from pathogenic streptococci in human blood serum comprising:

(a) adding at least an immunochemical euqivalent of a protein-A, said protein-A being able to bind non-specifically to said antibody to be determined and being bound to an insoluble carrier to human blood serum containing said antibody to be determined;
(b) adding to the mixture of step (a) at least an immunochemical equivalent of an esterase from pathogenic streptococci to form an esterase-anti-esterase antibody-insoluble protein-A complex;
(c) separating said complex from the mixture of step (b) by centrifugation; and
(d) measuring the activity of the esterase in said complex by using a substrate which is selected from the group consisting of S-acethylthiophenol, naphthylacetate and nitrophenylacetate.

7. A method for the quantitative determination of an antibody against an esterase from pathogenic streptococci in human blood comprising:

(a) adding simultaneously at least an immunochemical equivalent of an esterase from pathogenic streptococci and at least an immunochemical equivalent of a protein-A, said protein-A being able to bind non-specifically to said antibody to be determined and being bound to an insoluble carrier to human blood serum containing said antibody to be determined to form an esterase-anti-esterase antibody-insoluble protein-A complex;
(b) separating said complex from the mixture of step (a) by centrifugation; and
(c) measuring the activity of the esterase in said complex by using a substrate which is selected from S-acetylthiophenol, naphthylacetate and nitrophenylacetate.
Referenced Cited
U.S. Patent Documents
3790447 April 1974 HiratIa et al.
3791932 February 1974 Schuurs et al.
3932221 January 13, 1976 Pfleider
4012285 March 15, 1977 Pfleider et al.
4016043 April 5, 1977 Schuurs et al.
4166767 September 4, 1979 Kurooka et al.
4366242 December 28, 1982 Neumann et al.
Other references
  • Dainippon Pharmaceutical Co., "Reagents for Antibody Determination", Chem. Absts., vol. 95, No. 7, (1981) Absts. #57653f. Soudon et al., "A New Rapid and Reliable Test for the Demonstration of Streptococcal Anti-Exoenzyme Antibodies", Chem. Absts., vol. 86, No. 17 (1977) p. 368, Absts. No. 119048n. Hayano et al., "Repetitive Counterelectrophoresis on Agar Gel for the Immunological Identification of Esterases Produced by Strains of Lance Field's Group A B and C Streptococci", Infection and Immunology, vol. 15, No. 1, (1977), pp. 295-299. Forsgren et al., "Protein A from S. Aureus", J. Immunol, vol. 97, No. 6 (1966), pp. 822-827. Frohman et al., "Use of Protein A-Containing Streptococcus Aureus as an Immuno Adsorbent in Radio Immunonassays to Separate Antibody-Bound from Free Antigen", J. Lab. Clin. Med., vol. 93, No. 4 (1979), pp. 614-620. Shigeyo Shibazaki et al.; J. Clin. Lab. Inst. Reag., 6(4), 1037-1043 (1983) Abstract. Seiichi Kawakita et al.; Clin. Report, 17(9), 2994-3002 (1983) Abstract. Kunio Nakajima et al.; J. Pediatric Practice, 46(9), 1419-1438 (1983) Abstract. Yukiyoshi Nakamura et al.; J. Jap. Assoc. Infect. Dis., 57(9), 771-775 (1983) Abstract.
Patent History
Patent number: 4592995
Type: Grant
Filed: Nov 16, 1983
Date of Patent: Jun 3, 1986
Assignee: Dainippon Pharmaceutical Co., Ltd. (Osaka)
Inventors: Seiki Hayano (Omiya), Kanae Yokogawa (Nara), Shigeru Kurooka (Fujiidera)
Primary Examiner: Esther M. Kepplinger
Law Firm: Stevens, Davis, Miller & Mosher
Application Number: 6/552,014
Classifications
Current U.S. Class: 435/7; Packaged Device Or Kit (435/810); Carrier Is A Biological Cell Or Cell Fragment (436/519); Glass Or Silica (436/527); Polysaccharide, Carrier (e.g., Dextran, Etc.) (436/529); Protein A (436/828)
International Classification: G01N 33573; G01N 33569; G01N 33554; G01N 33548; G01N 33552;